CN107142229B - It is a kind of prevent and treat capsicum epidemic disease biocontrol actinomycetes bacterial strain and its application - Google Patents

It is a kind of prevent and treat capsicum epidemic disease biocontrol actinomycetes bacterial strain and its application Download PDF

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CN107142229B
CN107142229B CN201710256478.4A CN201710256478A CN107142229B CN 107142229 B CN107142229 B CN 107142229B CN 201710256478 A CN201710256478 A CN 201710256478A CN 107142229 B CN107142229 B CN 107142229B
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CN107142229A (en
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李本金
陈庆河
刘裴清
王荣波
翁启勇
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Abstract

The invention discloses a kind of biocontrol actinomycetes bacterial strains and its application method for preventing and treating capsicum epidemic disease, belong to crop disease control technical field.The biocontrol actinomycetes bacterial strain be scab streptomycete (Streptomyceseuropaeiscabiei) St19, preservation, deposit number CGMCCNO.8962 are registered in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 24th, 2014.The bacterial strain inhibits the growth of Phytophthora capsici by generating active material, shows preferable control efficiency to capsicum epidemic disease.Using when can be used in seedling stage of red pepper seedling stage or Adult plant pouring root.The microbial inoculum can also be mixed and made into bacterial manure use with organic fertilizer.Bacterial strain St19 of the present invention is obtained from capsicum rhizosphere soil, compatible with soil ecology harmony, nontoxic, no pathogenicity, therefore is had a good application prospect in the biological control of capsicum epidemic disease.

Description

It is a kind of prevent and treat capsicum epidemic disease biocontrol actinomycetes bacterial strain and its application
Technical field
The present invention relates to a kind of biocontrol actinomycetes bacterial strain for preventing and treating capsicum epidemic disease and its applications, belong to crop disease control Technical field.
Background technique
By Phytophthora capsici(Phytophthora capsici)Caused capsicum epidemic disease is a kind of destructiveness in capsicum production Soil-borne disease, have generation in each pepper producing area in the world at present.The disease not only can be by rainwater or irrigation water in the soil It propagates, causes large area dead seedling, can also be propagated by air-flow, caulis capsici of causing harm, leaf, flower, fruit, in suitable onset condition Under, disease breaks out cause disaster in a short time, host crop can be caused to have no harvest, and seriously affects China's Hot Pepper Industry Development, causes great Economic loss.
The prevention and treatment of capsicum epidemic disease at present is mainly or dependence cultivates disease-resistant variety and uses chemical agent, wherein cultivating disease-resistant Kind is prevention and treatment most economical, most effective, the simplest method of the disease, but since the easy variability of epidemic disease bacterium group causes very much Disease-resistant variety gradually loses original resistance, and it is very tired for being selected in a short time with horizontal antilemic new varieties Difficult.Although pesticide plays an important role to the development of agricultural, but with the development of society, with peace caused by chemical pesticide Complete and environmental problem is also increasingly prominent.Not only lack long-term validity, but also the dirt to food and environment using chemical pesticide Dye, influence potentially hazardous to human health, to non-target organism and lead to problems such as pathogen drug resistance generate, More and more queried.Therefore, the utilization of biological control especially antagonistic microbe is explored by common concern and attention Effective biological and ecological methods to prevent plant disease, pests, and erosion approach undoubtedly has great theory significance and practical value.
Actinomyces (Actinomycetes) are a kind of microorganisms with important economic value and practical value, and type is rich Richness distributes widely in nature.At present in the about 8000 kinds of bioactive substances found in microorganism, nearly 70% is It is generated by actinomyces, the antibiotic that especially streptomycete generates is widely used in agricultural production, such as Zhongshengmycin, avermectin Deng, all have pollution-free, noresidue, renewable, effect is lasting, it is with strong points, be difficult to harmful organism is made to develop drug resistance, easily In industrialization, at low cost the features such as, environmental benefit and economic benefit with higher, development prospect are extremely wide in agricultural production It is wealthy.Therefore, current agricultural scientific research is had become by screening and reaching efficient, environmentally friendly biological control purpose using Antagonistic Fungi One of hot spot, meet the sustainable development requirement of agricultural.
Summary of the invention
It is an object of the invention to: seriously occur for current capsicum epidemic disease, and area expands year by year, chemopreventive effects It is undesirable, while the problem of bring environmental pollution, provide an efficient prevention biocontrol actinomycetes and its application side of capsicum epidemic disease Method.
A kind of biocontrol actinomycetes bacterial strain and its application method for preventing and treating capsicum epidemic disease, the purpose of the present invention can pass through following skill Art scheme is realized:
A kind of biocontrol actinomycetes St19 preventing and treating capsicum epidemic disease, through morphologic observation, cultural characteristic observation and molecular biology Research be accredited as streptomycete (Streptomyces sp.), Chinese microorganism strain preservation is deposited in 24 months March in 2014 Administration committee's common micro-organisms center (CGMCC), address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science Institute of microbiology, institute, postcode 100101；Culture presevation number is CGMCC NO.8962.
Another object of the present invention is to provide a kind of biocontrol actinomycetes bacterial strain St19 using above-mentioned prevention and treatment capsicum epidemic disease The biocontrol agent of preparation, the biocontrol agent are fermented by the actinomycetes strain St19.
The present invention also provides the above-mentioned biocontrol actinomycetes bacterial strain St19 using prevention and treatment capsicum epidemic disease to prepare biocontrol agent Method, its step are as follows:
1) preparation of seed liquor: by the strain inoculated in Gause I fluid nutrient medium, 28 DEG C, 200 r/ Seed liquor is made after cultivating 2 d in min；
2) it ferments: seed liquor being inoculated in fermentation medium, fermentation condition are as follows: 6% vol of inoculum concentration, bottling amount 70mL 7.0,28 DEG C, 200 r/min, 5 d of fermentation time of/250mL, fermentation medium initial pH value, it is 10 that concentration, which is made,8CFU/ml St19 bacterial strain fermentation liquor；
3) preparation of bacteria-free filtrate: after 4000 r/min of fermentation liquid is centrifuged 5 min, with 0.45 μm of sterilizing miillpore filter Filtration sterilization obtains bacteria-free filtrate.
Wherein, the formula of fermentation medium described in step 2 are as follows: soluble starch 2%wt, glucose 1.5%wt, soybean powder 2%wt、MgSO4·7H2O 0.05%wt、K2HPO4 0.1%wt、NaCl 0.1%wt、CaCO30.2%wt, remaining is distilled water.
The application mode of above-mentioned biocontrol agent has following two:
1) it is used in seedling stage of red pepper seedling stage or Adult plant pouring root: being used continuously 2 ~ 3 times in seedling stage of red pepper seedling stage or Adult plant, every time It is spaced 7 ~ 10d；
2) bacterial manure use is mixed and made into organic fertilizer.
The biocontrol actinomycetes bacterial strain St19 of above-mentioned prevention and treatment capsicum epidemic disease can be applied in capsicum epidemic disease biological control.
The present invention has the advantages that
The biocontrol actinomycetes St19 that the present invention filters out can obviously inhibit the growth of Phytophthora capsici.Due to being biological system Agent, therefore not use bring a series of problems of chemical pesticide, can reduce farmland pollution, and can effectively prevent capsicum epidemic disease Disease.
Biocontrol actinomycetes of the present invention are isolated from capsicum rhizosphere soil, compatible with soil ecology harmony, are conducive to give full play to The advantage of bacterial strain.
Biocontrol actinomycetes strain culturing condition of the present invention is simple, easy to industrialized production, before having good development and application Scape.
Detailed description of the invention
Fig. 1 is bacterial strain St19 aerial hyphae and spore filament shapes (400 × under optical microscopy).
Fig. 2 is bacterial strain St19 hypha form (electron microscope 5000 ×).
Fig. 3 is plate fungistatic effect figure of the antagonistic strain St19 to Phytophthora capsici.
Specific embodiment
For a better understanding of the present invention, the present invention is further elaborated with reference to the accompanying drawings and examples, but not It is limitation of the present invention.Experimental methods as used in the following examples are conventional method unless otherwise specified.
The separation of one Antagonistic Actinomycetes of embodiment
Detailed process is as follows:
1, the acquisition of pedotheque
The rhizosphere soil of different 5 ~ 10 depths cm of capsicum plot, packing are acquired from Fujian Province, the town Dong Feng, the Jianou City village Kun Kou Laboratory is taken back after label, is separated after natural air drying.
2, the separation of actinomyces
It is separated using plate dilution method.10 g of pedotheque is weighed, is poured into equipped with small bead and 90 ml sterile waters Triangular flask in, shake 30 min after stands 5 minutes, successively dilution 10 times, be configured to 10 respectively-2、10-3、10-4、10-5、10-6 Suspension, each 0.1 ml of suspension for drawing various concentration is added to Gause I culture medium and (final concentration of 50 mg/L is added K2Cr2O7) on plate, even spread is placed on 28 DEG C of cultures observations, and the single colonie that picking is different after 5 ~ 7 days is crossed purifying. Bacterial strain after purification is transferred on Gause I slant medium and is cultivated, 4 DEG C save backup, and isolate to obtain 80 plants of unwrapping wire Bacterium.
The identification of two bacterial strain St19 of embodiment
1, morphological feature is observed
By bacterial strain St19 streak inoculation on Gause I culture medium, enter sterile cover slips, 28 DEG C of trainings with 45 DEG C of oblique cuttings After supporting 10 d, coverslip is taken out in the morphological feature (Fig. 1 and Fig. 2) of optical microscopy and observed under electron microscope mycelia.
2, cultural characteristic is observed
The 8 kinds of culture mediums recommended using " streptomycete identification handbook ", bacterial strain St19 is observed after 28 DEG C of 7 ~ 10d of culture Thalli growth situation, record aerial hyphae, substrate mycelium color and whether generate soluble pigment.Bacterial strain St19 is in Gao Shi No.1 culture medium examines Bake culture medium, starch ammonium culture medium, PDA culture medium, glucose yeast cream culture medium and oat medium Upper well-grown, substrate mycelium and aerial hyphae develops well；And in kirschner No.1 culture medium and glucose asparagine culture Poor, the yellow colour fading of generation or brown colour fading soluble pigment (table 1) on Gause I culture medium and PDA culture medium are grown on base. According to the above morphometric analysis, compare streptomycete identification handbook, by St19 be accredited as streptomycete (Streptomyces sp.)。
Cultural characteristic of the 1 bacterial strain St19 of table in different culture medium
3,16S rDNA sequence is analyzed
After genome extraction kit extracts bacterial strain St19 genomic DNA, using universal primer F:5'- AGAGTTTGATCCTGGCTCAG-3' and R:5'-GGTTACCTTGTTACGACTT-3', the PCR for carrying out 16S rDNA expand Increase.Recycling pcr amplification product obtains St19-16S rDNA, and after connection, conversion, identification, positive colony send raw work biology work Nucleotide sequence information is sequenced to obtain in journey (Shanghai) Co., Ltd., and sequence is 1548 bp.Gained sequence is submitted to GenBank database carries out BLAST analyses and comparison, and the bacterial strain that discovery reaches 99% with St19 homology belongs to streptomyces, Combining form feature and cultural characteristic, finally by bacterial strain St19 be accredited as streptomycete (Streptomyces sp.)。
The optimization of three St19 strain fermentation process of embodiment
Above-mentioned bacterial strains are inoculated in Gause I fluid nutrient medium, 28 DEG C, 200 r/min, after cultivating 2 d, kind is made Sub- liquid, is inoculated in fermentation medium.Using Phytophthora capsici as target bacterium, pass through screening to carbon source, nitrogen source and nutritional condition After single factor experiment and orthogonal test, the best fermentating formula of the bacterial strain is obtained are as follows: soluble starch 2%wt, glucose 1.5% Wt, soybean powder 2%wt, MgSO4·7H2O 0.05%wt、K2HPO4 0.1%wt、NaCl 0.1%wt、CaCO30.2%wt, remaining is Distilled water.Optimal conditions of fermentation are as follows: 6% vol of inoculum concentration, bottling amount 70 mL/250 mL, fermentation medium initial pH value 7.0, 28 DEG C, 200 r/min, 5 d of fermentation time, it is 10 that concentration, which is made,8 The St19 bacterial strain fermentation liquor of CFU/ml.By fermentation liquid 4000 After r/min is centrifuged 5 min, with 0.45 μm of sterilizing filtering with microporous membrane degerming, bacteria-free filtrate is obtained.
The measurement of example IV bacterial strain St19 antagonism
Using plate opposite culture method, bacterial strain St19 is made into antagonism measurement to Phytophthora capsici, it is flat in rye culture medium first 5 cm Phytophthora capsici bacteria cakes are accessed in plate center, when Phytophthora capsici bacterium colony it is long to 3 cm when, put at bacterium colony 4 weeks and connect bacterial strain St19, Measurement bacterial strain St19 compares the antibacterial bandwidth of Phytophthora capsici not connect Antagonistic Fungi after cultivating 5 d at 25 DEG C, the results showed that Bacterial strain St19 has preferable antagonism (Fig. 3) to Phytophthora capsici.
Five bacterial strain St19 fermentation liquid potting efficiency test of embodiment
The eggplant door pimento seedling of 4 ~ 6 leaf phases is chosen, every young plant pours 50 ml St19 bacterial strain fermentation liquors (108CFU/ml). Pouring root is inoculated with Phytophthora Capsici Zoospores suspension (5 × 10 after 24 h3A/mL), every plant of 20 ml.Inoculation poured one after 4 days again Secondary 100 ml St19 bacterial strain fermentation liquor.It is compareed using 72% gram of dew wettable powder, 600 times of liquid irrigating root processing as pesticide, with fermentation The plant of culture medium and clear water processing is as blank control.10 young plants of each processing, 3 repetitions of every processing.After the onset daily Incidence is investigated, disease incidence and disease index are counted, until clear water control disease incidence terminates to test when reaching 80% or more.
Disease grade standard is as follows:
0 grade: disease-free；
1 grade: the slight blackening of seedling rhizome portion, blade is not wilted or restorability is wilted；
3 grades: seedling rhizome portion 1 ~ 2cm of blackening, blade irrecoverability is wilted, and lower blade, which occasionally has, to fall off；
5 grades: 2 cm or more of seedling rhizome portion blackening, blade irrecoverability is wilted or leaf abscission is obvious；
7 grades: seedling rhizome portion blackening is hung contracting, except growing point outer leafs all fall off or plant is wilted；
9 grades: plant is withered.
Disease index (%)=[the ∑ disease series × disease grade plant number)/(highest disease series × total plant number)] × 100%
Control efficiency (%)=[(blank control disease index-processing disease index)/blank control disease index] × 100%
Experimental result is shown in Table 2, should be the result shows that can significantly reduce the disease of capsicum epidemic disease for examination actinomyces St19 fermentation liquid Feelings index, control efficiency are up to 66.85%, are just slightly below the control efficiency of 72% gram of chemical pesticide dew 600 times of liquid of wettable powder 71.06%.In addition it can also be seen that the disease index of fermentation medium processing is slightly below the state of an illness of clear water control treatment from table Index, but the difference between the two is not significant.Greenhouse pot culture is the experiment results show that St19 bacterial strain can effectively prevent capsicum epidemic disease, performance Good application potential out.
Control efficiency of the 2 actinomyces St19 fermentation liquid of table to capsicum epidemic disease
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Inst. of Plant Protection, fujian Academy of Agricultural Science
<120>a kind of biocontrol actinomycetes bacterial strain for preventing and treating capsicum epidemic disease and its application
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> F
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213> R
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 1548
<212> DNA
<213> St19-16S rDNA
<400> 3
agcttgcatg cctgcaggtc gacgattaga gtttgatcct ggctcaggac gaacgctggc 60
ggcgtgctta acacatgcaa gtcgaacgat gaaccacttc ggtggggatt agtggcgaac 120
gggtgagtaa cacgtgggca atctgccctt cactctggga caagccctgg aaacggggtc 180
taataccgga tacaacactc tcgggcatcc gatgagtgtg gaaagctccg gcggtgaagg 240
atgagcccgc ggcctatcag cttgttggtg aggtaacggc tcaccaaggc gacgacgggt 300
agccggcctg agagggcgac cggccacact gggactgaga cacggcccag actcctacgg 360
gaggcagcag tggggaatat tgcacaatgg gcgaaagcct gatgcagcga cgccgcgtga 420
gggatgacgg ccttcgggtt gtaaacctct ttcagcaggg aagaagcgaa agtgacggta 480
cctgcagaag aagcgccggc taactacgtg ccagcagccg cggtaatacg tagggcgcga 540
gcgttgtccg gaattattgg gcgtaaagag ctcgtaggcg gtctgtcgcg tcgaatgtga 600
aagcccgggg cttaaccccg ggtctgcatt cgatacgggc agactagagt gtggtagggg 660
agatcggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac accggtggcg 720
aaggcggatc tctgggccat tactgacgct gaggagcgaa agcgtgggga gcgaacagga 780
ttagataccc tggtagtcca cgccgtaaac ggtgggaact aggtgttggc gacattccac 840
gtcgtcggtg ccgcagctaa cgcattaagt tccccgcctg gggagtacgg ccgcaaggct 900
aaaactcaaa ggaattgacg ggggcccgca caagcagcgg agcatgtggc ttaattcgac 960
gcaacgcgaa gaaccttacc aaggcttgac atacaccgga aacggccaga gatggtcgcc 1020
cccttgtggt cggtgtacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaacccttg ttctgtgttg ccagcatgcc cttcggggtg 1140
atggggactc acaggagact gccggggtca actcggagga aggtggggac gacgtcaagt 1200
catcatgccc cttatgtctt gggctgcaca cgtgctacaa tggcaggtac aatgagctgc 1260
gaagccgtga ggcggagcga atctcaaaaa gcctgtctca gttcggattg gggtctgcaa 1320
ctcgacccca tgaagtcgga gttgctagta atcgcagatc agcagtgctg cggtgaatac 1380
gttcccgggc cttgtacaca ccgcccgtca cgtcacgaaa gtcggtaaca cccgaagccg 1440
gtggcccaac cccttgtggg agggagctgt cgaaggtggg actggcgatt gggacgaagt 1500
cgtaacaagg taccaatctc tagaggatcc ccgggtaccg agctcgaa 1548

Claims (3)

1. a kind of biocontrol agent for the biocontrol actinomycetes bacterial strain St19 preparation for preventing and treating capsicum epidemic disease, which is characterized in that the bacterium Strain be streptomycete (Streptomyces sp.) St19, it is commonly micro- to be preserved in China Committee for Culture Collection of Microorganisms Bio-Centers, the deposit date is on March 24th, 2014, deposit number was CGMCCNO. 8962；
The biocontrol agent is fermented by the actinomycetes strain St19；Preparation method is as follows:
1) preparation of seed liquor: by the strain inoculated in Gause I fluid nutrient medium, 28 DEG C, 200 r/min, training After supporting 2 d, seed liquor is made；
2) it ferments: seed liquor being inoculated in fermentation medium, fermentation condition are as follows: 6% vol of inoculum concentration, bottling amount 70mL/ 7.0,28 DEG C, 200 r/min, 5 d of fermentation time of 250mL, fermentation medium initial pH value, it is 10 that concentration, which is made,8 CFU/ml's St19 bacterial strain fermentation liquor；
3) preparation of bacteria-free filtrate: after 4000 r/min of fermentation liquid is centrifuged 5 min, with 0.45 μm of sterilizing filtering with microporous membrane Degerming, obtaining bacteria-free filtrate is the biocontrol agent；
The formula of fermentation medium described in step 2 are as follows: 2% wt of soluble starch, 1.5% wt of glucose, 2% wt of soybean powder, MgSO4·7H2O 0.05% wt、K2HPO4 0.1% wt、NaCl 0.1% wt、CaCO30.2% wt, remaining is distilled water.
2. a kind of biocontrol agent of biocontrol actinomycetes St19 preparation using prevention and treatment capsicum epidemic disease according to claim 1, It is characterized in that, its application mode has following two:
1) it uses: being used continuously 2 ~ 3 times in seedling stage of red pepper seedling stage or Adult plant, every minor tick in seedling stage of red pepper seedling stage or Adult plant pouring root 7~10 d；
2) bacterial manure use is mixed and made into organic fertilizer.
3. using the biocontrol actinomycetes bacterial strain St19 of prevention and treatment capsicum epidemic disease described in claim 1 in capsicum epidemic disease biological control Using.
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